Volume 48, Issue 3, Pages 343-352 (November 2012) DNA Damage-Induced Primordial Follicle Oocyte Apoptosis and Loss of Fertility Require TAp63-Mediated Induction of Puma and Noxa  Jeffrey B. Kerr, Karla J. Hutt, Ewa M. Michalak, Michele Cook, Cassandra J. Vandenberg, Seng H. Liew, Philippe Bouillet, Alea Mills, Clare L. Scott, Jock K. Findlay, Andreas Strasser  Molecular Cell  Volume 48, Issue 3, Pages 343-352 (November 2012) DOI: 10.1016/j.molcel.2012.08.017 Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 1 Expression of Puma mRNA, Noxa mRNA, and PUMA Protein in Primordial Follicle Oocytes after γ-Irradiation-Induced DNA Damage (A) Ovaries were harvested from PN5 WT, Puma−/− (negative control), Noxa−/− (negative control), Trp53−/−, and TAp63 mutant mice at 0 (untreated control) and 3 hr after whole-body γ-irradiation (0.45 Gy). In situ hybridization (top panel) was performed with anti-sense probes for Puma and Noxa. Puma and Noxa mRNA was detected in primordial follicle oocytes from WT and Trp53−/− mice but not in those from TAp63 mutant mice, 3 hr after γ-irradiation. Arrows indicate positively stained primordial follicle oocytes (dark purple/brown staining). Control experiments, including in situ hybridization of sections from Puma−/− ovaries with Puma antisense probes and in situ hybridization of sections from Noxa−/− ovaries with Noxa antisense probes, are shown in Figures S1B and S1E. (B) PUMA antibody immunofluorescent staining (bottom panel; green) in WT and Puma−/− (negative control) primordial follicle oocytes at 6 hr after γ-irradiation. Scale bars represent 20 μm. See also Figure S1. Molecular Cell 2012 48, 343-352DOI: (10.1016/j.molcel.2012.08.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 2 Detection of DNA Damage and Apoptosis in Primordial Follicle Oocytes after γ-Irradiation (A) DNA double-strand breaks were analyzed in oocytes from untreated PN5 WT, Puma-/- and Puma−/−Noxa−/− primordial follicles and 3 hr after exposure to whole-body γ-irradiation at the lower (0.45 Gy) and higher (4.5 Gy) doses by immunofluorescent staining with antibody to γ-H2AX (red). γ-H2AX foci were resolved in Puma−/− and Puma−/−Noxa−/− primordial follicle oocytes within 5 days of γ-irradiation-induced DNA damage (no primordial follicles survived in WT mice at this time point). Even at the higher dose of γ-irradiation (4.5 Gy), evidence of prior DNA damage could no longer be detected after 5 days in most oocytes, with only a few Puma-/- and Puma−/−Noxa−/− oocytes exhibiting one or two γ-H2AX foci. Arrowheads indicate the nuclei of primordial follicle oocytes. (B) γ-irradiation-induced DNA damage was quantified by counting the numbers of γ-H2AX foci within the nuclei of WT, Puma−/−, Noxa−/−, and Puma−/−Noxa−/− primordial follicles 3 hr after exposure to whole-body γ-irradiation (0.45 Gy). No significant differences in the numbers of foci were observed between females of the different genotypes (p > 0.05). (C) The numbers of γ-H2AX foci within the nuclei of WT primordial follicles 3 hr after exposure to whole-body γ-irradiation at the lower (0.45 Gy) and higher (4.5 Gy) dose were quantified. ∗∗∗p < 0.001. (D) The numbers of γ-H2AX foci within the nuclei of WT and Puma−/−Noxa−/− primordial follicles 3 hr after exposure to the higher dose of whole-body γ-irradiation (4.5 Gy) were quantified. No significant difference was observed in the numbers of foci between the different genotypes (p > 0.05). (E) The numbers of γ-H2AX foci within the nuclei of Puma−/−Noxa−/− primordial follicles were quantified as in (C), i.e., 3 hr after exposure to whole-body γ-irradiation at the lower (0.45 Gy) and higher (4.5 Gy) dose. Data represent the means ± SEM, n = 3–5 mice. ∗∗∗p < 0.001. n = 50–100 primordial follicles for all. Scale bars represent 20 μm. See also Figure S2. Molecular Cell 2012 48, 343-352DOI: (10.1016/j.molcel.2012.08.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 3 Loss of PUMA and Combined Loss of PUMA and NOXA Rescue Primordial Follicle Oocytes from DNA Damage-Induced Apoptosis (A) Quantification (means ± SEM) of follicles from mice of the indicated genotypes either exposed to whole-body γ-irradiation (0.45 or 4.5 Gy) or untreated at PN5 and analyzed at PN10. The percent primordial follicle survival (compared to untreated) is shown above each bar. n = 3–8 animals/genotype. For comparison with WT, ∗p < 0.05 and ∗∗p < 0.001. (B) MSY2 (the germ cell-specific Y-box cytoplasmic protein marker, MSY2, which stains all follicle types) antibody staining of ovaries of PN10 WT, Noxa−/−, Puma−/−, Puma−/−Noxa−/−, and Trp53−/− mice 5 days after exposure to 0.45 or 4.5 Gy γ-irradiation or no treatment (control). Arrows indicate intrinsically DNA damage-resistant growing (postprimordial) follicles. Black arrowheads indicate primordial follicles. The scale bar represents 200 μm. See also Figure S3. Molecular Cell 2012 48, 343-352DOI: (10.1016/j.molcel.2012.08.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 4 Loss of PUMA or Combined Loss of PUMA and NOXA Rescues GCNA-Positive Primordial Follicle Oocytes from DNA Damage-Induced Apoptosis and Preserve Fertility in γ-Irradiated Mice (A) Anti-GCNA (the nuclear marker, germ cell-specific antigen that stains only primordial follicles) antibody staining of ovaries of PN10 WT, Trp53−/−, Noxa−/−, Puma−/−, and Puma−/−Noxa−/− mice 5 days after exposure to 0.45 Gy or 4.5 Gy γ-irradiation or no treatment (control). Arrows indicate intrinsically resistant growing follicles. Black arrowheads indicate primordial follicles. White arrowheads indicate primordial follicle remnants. The scale bar represents 20 μm. (B) Female WT, Puma−/−, and Puma−/−Noxa−/− mice (PN5) were left untreated or exposed to whole-body γ-irradiation (0.45 Gy), and after at least 7 weeks of age (i.e., 45 days post γ-irradiation) breeding trials were commenced. The numbers of pups from first and second litters from matings to WT C57BL/6 proven male breeders are shown. ∗∗p value < 0.001 for Puma−/− γ-irradiated versus WT γ-irradiated or Puma−/−Noxa−/− γ-irradiated versus WT γ-irradiated, for all categories. All other comparisons were not significant. Second litters from Puma−/− and Puma−/−Noxa−/− females were included to show protection was not limited to the first litter. Data represent the means ± SD, n = 6–12 litters. Refer also to Tables 1 and 2. Molecular Cell 2012 48, 343-352DOI: (10.1016/j.molcel.2012.08.017) Copyright © 2012 Elsevier Inc. Terms and Conditions

Figure 5 PUMA and NOXA Are Critical for TAp63-Induced Apoptosis DNA damage leads to activation of TAp63, resulting in transcriptional induction of Puma and Noxa (which may be direct or indirect), with subsequent binding of PUMA and NOXA proteins to prosurvival BCL-2 family members (BCL-2, BCL-XL, BCL-W, MCL-1, or A1) and possibly also direct binding of PUMA to BAX/BAK. This causes activation of BAX and BAK, resulting in apoptotic cell death. Molecular Cell 2012 48, 343-352DOI: (10.1016/j.molcel.2012.08.017) Copyright © 2012 Elsevier Inc. Terms and Conditions